Validation of digital PCR as a complementary tool to MS2 plaque assay for virucidal activity assessment.

INTRODUCTION: Bacteriophage MS2 is widely applied as a surrogate for pathogenic non-enveloped RNA viruses that are difficult to culture. However, standardized approaches for evaluating disinfectant efficacy using MS2 remain limited. This study aimed to validate dPCR as a rapid quantitative complement to the previously established MS2 plaque assay, assessing their concordance in disinfectant efficacy testing using a previously established double-layer protocol. METHODS: A previously established double-layer MS2 plaque assay was employed. Disinfectant efficacy was tested against two widely used agents-70% ethanol and 60 ppm hypochlorous acid (HOCl)-according to the Korean Disinfectant Efficacy Test Guideline. Viral reduction was determined by plaque counts and further quantified by dPCR following RNase treatment to eliminate free RNA. Agreement between the established plaque assay and dPCR was evaluated using regression and Bland-Altman analyses. RESULTS: Treatment with 70% ethanol yielded only partial inactivation, with a 2.43-log10 reduction, below the ≥4-log10 benchmark for virucidal efficacy. In contrast, 60 ppm HOCl achieved a 4.38-log10 reduction, confirming effective MS2 inactivation. The dPCR counts correlated strongly with plaque counts (R = 0.999), and Bland-Altman analysis demonstrated excellent agreement within 95% confidence limits, supporting dPCR as a practical alternative to plaque assays. However, the reliability of dPCR as a proxy at low viral concentrations near the limit of detection (<102 PFU/mL) requires further validation. CONCLUSION: This study successfully validated dPCR as a rapid quantitative complement to the previously established MS2 double-layer plaque assay, demonstrating its utility in disinfectant efficacy testing. The findings confirm ethanol's limited virucidal capacity against MS2 and HOCl's effectiveness at low concentrations. These results establish dPCR as a promising molecular tool that complements plaque assay results for comprehensive viral surrogate evaluation in disinfection studies.